A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors.
shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we descr...
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doaj-7026b03a2731462481f9b6f5badc98242020-11-24T21:50:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011310e020558510.1371/journal.pone.0205585A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors.Severin FinkLaurens ZugelderBernhard RothEvelyn BrandtSylvain MelocheZsuzsanna IzsvákRalf C BargouThorsten StühmershRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids. The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level. We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway. The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models.http://europepmc.org/articles/PMC6195277?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Severin Fink Laurens Zugelder Bernhard Roth Evelyn Brandt Sylvain Meloche Zsuzsanna Izsvák Ralf C Bargou Thorsten Stühmer |
spellingShingle |
Severin Fink Laurens Zugelder Bernhard Roth Evelyn Brandt Sylvain Meloche Zsuzsanna Izsvák Ralf C Bargou Thorsten Stühmer A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. PLoS ONE |
author_facet |
Severin Fink Laurens Zugelder Bernhard Roth Evelyn Brandt Sylvain Meloche Zsuzsanna Izsvák Ralf C Bargou Thorsten Stühmer |
author_sort |
Severin Fink |
title |
A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. |
title_short |
A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. |
title_full |
A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. |
title_fullStr |
A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. |
title_full_unstemmed |
A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors. |
title_sort |
simple approach for multi-targeted shrna-mediated inducible knockdowns using sleeping beauty vectors. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2018-01-01 |
description |
shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids. The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level. We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway. The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models. |
url |
http://europepmc.org/articles/PMC6195277?pdf=render |
work_keys_str_mv |
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